Interferon gamma (IFNG) is a cytokine produced by T-lymphocytes and natural killer cells and exists as a homodimer of two noncovalently bound polypeptide subunits. The mature form of each monomer comprises 143 amino acid residues (shown in SEQ ID NO:1) and the precursor form thereof, including the signal sequence, comprises 166 amino acid residues (shown in SEQ ID NO:2).
Each subunit has two potential N-glycosylation sites (Aggarwal et al., Human Cytokines, Blackwell Scientific Publications, 1992) at positions 25 and 97. Depending on the degree of glycosylation the molecular weight of IFNG in dimer form is 34-50 kDa (Farrar et al., Ann. Rev. Immunol, 1993, 11:571-611).
The primary sequence of wild-type human IFNG (huIFNG) was reported by Gray et al. (Nature 298:859-863, 1982), Taya et al. (EMBO J. 1:953-958, 1982), Devos et al. (Nucleic Acids Res. 10:2487-2501, 1982) and Rinderknecht et al. (J. Biol. Chem. 259:6790-6797, 1984), and in EP 77670, EP 89676 and EP 110044.
Experimental 3D structures of huIFNG determined by X-ray crystallography have been reported by Ealick et al. Science 252:698-702 (1991) who reported the C-alpha trace of an IFNG homodimer. Walter et al. Nature 376:230-235 (1995) disclosed the structure of an IFNG homodimer in complex with two molecules of a soluble form of the IFNG receptor. The coordinates of this structure, however, have never been made publicly available. Thiel et al. Structure 8:927-936 (2000) showed the structure of an IFNG homodimer in complex with two molecules of a soluble form of the IFNG receptor having a third molecule of the receptor in the structure not making interactions with the IFNG homodimer.
Various naturally-occurring or mutated forms of the IFNG subunit polypeptides have been reported, including one comprising a Cys-Tyr-Cys N-terminal amino acid sequence (positions (−3)-(−1) relative to SEQ ID NO:1), one comprising an N-terminal methionine (position −1 relative to SEQ ID NO:1), and various C-terminally truncated forms comprising 127-134 amino acid residues. It is known that 1-15 amino acid residues may be deleted from the C-terminus without abolishing IFNG activity of the molecule. Furthermore, heterogeneity of the huIFNG C-terminus was described by Pan et al. (Eur. J. Biochem. 166:145-149, 1987).
Glycosylation variation in huIFNG has been reported by Curling et al. (Biochem. J. 272 :333-337, 1990) and Hooker et al., (J. of Interferon and Cytokine Research, 1998, 18: 287-295).
Polymer-modification of huIFNG was reported by Kita et al. (Drug Des. Deliv. 6 :157-167, 1990), and in EP 236987 and U.S. Pat. No. 5,109,120.
WO 99/03887 discloses PEGylated variants of polypeptides belonging to the growth hormone superfamily, wherein a non-essential amino acid residue located in a specified region of the polypeptide has been replaced by a cysteine residue. IFNG is mentioned as one example of a member of the growth hormone super family, but modification thereof is not discussed in any detail.
WO 01/36001 discloses novel IFNG conjugates comprising a non-polypeptide moiety attached to an IFNG polypeptide which have been modified by introduction and/or deletion of attachment sites for such non-polypeptide moieties, e.g. PEG and glycosylation sites. These molecules have improved properties, such as improved half-life and/or increased bioavailablity.
IFNG has been suggested for treatment of interstitial lung diseases (also known as Interstitial Pulmonary Fibrosis (IPF) (Ziesche et al. (N. Engl. J. Med. 341:1264-1269, 1999 and Chest 110:Suppl:25S, 1996) and EP 0 795 332) for which purpose IFNG can be used in combination with prednisolone. In addition to IPF, granulomatous diseases (Bolinger et al, Clinical Pharmacy, 1992, 11:834-850), certain mycobacterial infections (N. Engl. J. Med. 330:1348-1355, 1994), kidney cancer (J. Urol. 152:841-845, 1994), osteopetrosis (N. Engl. J. Med. 332:1594-1599, 1995), scleroderma (J. Rheumatol. 23:654-658, 1996), hepatitis B (Hepatogastroenterology 45:2282-2294, 1998), hepatitis C (Int. Hepatol. Communic. 6:264-273, 1997), septic shock (Nature Medicine 3:678-681, 1997), and rheumatoid arthritis may be treated with IFNG. Furthermore, IFNG is presently being clinically evaluated for treatment of ovarian cancer, liver fibrosis, asthma and lymphoma.
As a pharmaceutical compound huIFNG is used with a certain success, above all, against some viral infections and tumors. huIFNG is usually applicable via parenteral, preferably via subcutaneous, injection. Maximum serum concentrations have been found after seven hours. It has been reported that the half-life in plasma is 30 minutes after intravenous administration. For this reason efficient treatment with huIFNG involves frequent injections.
The main adverse effects consist of fever, chills, sweating, headache, myalgia and drowsiness. These effects are associated with injecting huIFNG and are observed within the first hours after injection. Rare side effects are local pain and erythema, elevation of liver enzymes, reversible granulo- and thrombopenia and cardiotoxicity.
It is known that when IFNG is produced in mammalian cell lines a heterogenous population of IFNG polypeptides is obtained due to C-terminal truncation of the IFNG polypeptide (reviewed in Lundell et al. Pharmac. Ther. 64, 1-21, 1994). Clearly, this constitutes a severe problem in that valuable polypeptide material is lost and, further, it is necessary to carry out time-consuming and cumbersome purification in order to obtain a homogenous population of IFNG polypeptides having the desired length. Most likely, this truncation is effected by endo- and/or exoprotease activity present in the cell. The present inventors have solved the above-mentioned problem by performing selected modifications in the C-terminal part of the IFNG polypeptide. By performing such modifications C-terminal truncation is avoided and, consequently, the truncation can be controlled and homogenous populations of full-length IFNG polypeptides can be obtained.
Thus, it is an object of the present invention to provide novel full-length IFNG polypeptides, which are not prone to C-terminal truncation during production or storage.